1. Field
This disclosure is concerned with the preparation of an immunogenic protein material from Streptococcus equi bacteria using an enzymatic digestion and detergent treatment and use of the material as a vaccine against Strep. equi infection in equines.
2. Prior Art
Streptococcus equi is classified as a Lancefield Group C Streptococcus. See, for example, Bergey's Manual of Determinitive Bacteriology (8th Ed.), p. 498 (1974). It is recognized as the causative agent of a severe respiratory disease of horses referred to as "Strangles". The disease is endemic in most parts of the world and epidemic in the United States. Race and show horses are particularly susceptible to repeated infections due to the stress of travel and exposure to new contacts. The disease begins with a mucopurulent nasal discharge, temperatures of 103.degree.-106.degree. F., and severe inflammation of the upper respiratory mucosa. It finally progresses to lymphadenitis and abscess formation which is sometimes severe enough to restrict air intake and cause suffocation of the animal. Strangles results in extensive loss of condition (loss of weight) as it often runs a course of 4-6 weeks.
Because of the debilatating and in some cases lethal effects of Strep. equi infections in horses, attempts have been made over the years to prepare Strep. equi vaccines which could be used for active immunization purposes. Unfortunately, Strep. equi preparations have been noted for their affinity for dermal tissue, producing severe swelling and even abscess formation at the injection site. These known reactivities have tended to discourage the development and/or commercial use of immunizing Strep. equi products. Two commercially available Strangles vaccines do exist, however. The first commercial product was a whole culture, chemically inactivated Strep. equi preparation (supplied by Ft. Dodge Corporation). The second commercial product was a cell free M-protein vaccine (available from Burroughs Wellcome Co.) described as "a concentrated, aluminum hydroxide-absorbed suspension of purified antigens derived from Strep. equi". The method by which this vaccine is prepared is thought to be described in U.S. Pat. No. 3,793,150 and U.S. Pat. No. 3,852,420. The purification of such "M-like proteins" from Strep. equi is also described in an article by J. B. Woolcock, Infect. and Immun., July 1974, p. 116-122. As used herein, the expression "M-like protein" means the immunogenic protein(s) of the Strep. equi organism which appears similar in molecular weight and activity to the M-protein of group A streptococci.
The theory that a protein on the cell wall of the Strep. equi organism, referred to as an M-like protein, is the antigenic portion of the bacteria has been discussed in articles by S. K. Srivastava and D. A. Barnum in the Can. J. Comp. Med., Vol. 46, p. 51-56, 1982 and in the Am. J. Vet. Res., Vol. 44, p. 41-45, 1983 and in articles by E. D. Erickson and N. L. Norcross in the Can. J. Comp. Med., Vol. 39, p. 110-115, 1975. In all previous work, this M-like protein was extracted from the Strep. equi organism by subjecting the organism to low pH conditions (pH2) and high temperatures (95.degree.-100.degree. C.) for a given time (10-15 minutes). This has been referred to as a "heat extraction" method of preparing the M-like protein. The protein precipitates under these conditions and is solubilized by raising the pH of the solution to pH 7 or above.
We now have developed an improved method of removing the M-like protein from Strep. equi organisms, details of which are described herein.
We have now found that the antigenic M-like protein can be efficaciously removed from a Strep. equi culture in a two step process using lytic enzyme digestion followed by treatment with an anionic detergent and that this extract can be used to prepare a vaccine effective in immunizing horses against infection by Strep. equi. The potency of this antigen preparation has been determined using the method stated in above-cited patent application Ser. No. 454,906 now, U.S. Pat. No. 4,529,581, entitled, Determining Potency of Streptococcal Preparations, and has been confirmed in a horse challenge study, described herein.